Effects of a novel soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), were characterized on guanylyl cyclase activity in cytosolic fraction of COS-7 cells overexpressing the α₁ and β₁ subunits of the rat soluble enzyme. ODQ was a noncompetitive inhibitor of soluble guanylyl cyclase with respect to Mn²⁺ or Mn²⁺-GTP and was a mixed competitive/noncompetitive inhibitor with respect to nitric oxide (NO) donation. ODQ (10 μmol/L) reduced deta nonoate-stimulated cGMP production in COS-7 cells overexpressing soluble guanylyl cyclase and in rat aortic vascular smooth muscle cells. ODQ did not inhibit particulate forms of the enzyme rat guanylyl cyclase-A, -B, or -C, did not block NO synthase, and did not auto-oxidize deta nonoate-donated NO in the presence of cells at physiological pH. Therefore, ODQ is a selective inhibitor of soluble guanylyl cyclase. Using ODQ in isolated aortic ring preparations, we tested the hypothesis that soluble guanylyl cyclase mediates vasorelaxant activity associated with NO. Phenylephrine (100 nmol/L)-precontracted, isolated rat aortas were relaxed in a concentration-dependent manner by deta nonoate (0.01 to 100 μmol/L) and nitroglycerin (0.01 to 300 μmol/L). ODQ (10 μmol/L) attenuated deta nonoate- and nitroglycerin-mediated relaxation of contracted aortas. ODQ had no effect on natriuretic peptide-, 8-bromo-cGMP-, isoproterenol-, or bimak-alim-mediated aortic relaxation. These results support the hypothesis that soluble guanylyl cyclase mediates vasorelaxant activity associated with nitric oxide.